3 replies to this topic

[beth]

Hi there,

I want to look at the methylation status of Foxp3, which is located on the X chromosome. All papers I have read say that they only look at male patients/mice, to avoid possible artifacts due to X linked inactivation in females. My problem is that the disease I am studying has a large female preponderance (>95% of my samples are female) and so I really need to measure methylation in female cells. Does anyone know of any way that I can overcome this problem? Or even how I can can calculate some sort of probability of how much the inactivation may impact on my results so I can at least defend myself when I come to publish? I am thinking that if at least look at quite a few patients, and compare to the same number of female controls, that I can make some sort of conclusions...?

Thanks for any advice you can give,

Beth

p.s. The method I am using is the SABiosciences (Qiagen) EpiTect Methyl qPCR Primer Assay for Human FOXP3

[pcrman]

There are many strategies available for your study and they are called allele-specific DNA methylation analysis. If you search pubmed, you will be able to find some. Usually the alleles are differentiated by SNPs.

[methylnick]

does foxp3 fall within the known inactivation domains? is it monoallelically expressed in females? without knowing too much about it, it may not be too much of an issue.

[billyrosewood]

Hi - I feel a fraud for commenting on this as I have fairly minimal experience but we are trying to look at this too. Every other paper I've seen has purely multiplied the demethylated fraction (be it by bisulfite sequencing or MSP) by 2 in females to account for the other methylated, inactivated chromosome. We are attempting to use a modification of Kubuta et al to determine whether there skewing is a major factor but my understanding is that skewed X inactivation is less pronounced in lymphocytes than other cell types.